Project Summary Current therapies for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are unsatisfactory. We and others discovered that components of the pre-mRNA splicing complex are recurrently mutated in patients with MDS and AML and induce alterations in RNA splicing. Small molecule modulators of this complex (i.e., splicing modulators) have been developed by our collaborators and others. We recently reported that expression of mutant splicing factors or pharmacologic perturbation of splicing with splicing modulators increase the abundance of R loops, which are structures containing DNA:RNA hybrids and displaced single-strand DNA. R loops trigger an ATR-dependent DNA damage checkpoint response that mediates resolution of the R loop to protect cells from genomic instability and cell death. Our preliminary preclinical data suggest that spliceosome mutant cells are more sensitive to ATR inhibition than wild-type cells. We hypothesize that splicing factor mutations create a vulnerability to ATR inhibition (ATRi) that can be exploited for the development of novel therapeutic strategies when used alone or in combination with splicing modulator drugs. We will test this hypothesis in a phase II clinical trial of an ATRi in patients with myeloid malignancies and using well-defined in vitro and in vivo models. In Specific Aim 1, we will conduct a phase II trial of ATR inhibition in relapsed/refractory MDS and chronic myelomonocytic leukemia (CMML) and determine the molecular basis of sensitivity and resistance to this novel therapy. We predict that patients with spliceosome mutations will have an increased response rate to ATRi compared to patients without a splicing factor mutation. Correlative studies will assess pharmacodynamic endpoints and examine the mechanisms of drug sensitivity and resistance, if it occurs. In Specific Aim 2, we will test the efficacy of ATRi in combination with splicing modulators in models of spliceosome-mutant MDS. Preliminary evidence suggests that exposure of splicing factor mutant expressing hematopoietic cells to small molecule modulators of the spliceosome exacerbates R loop formation and increases sensitivity of cells to ATRi. We will use genetically-defined cellular and mouse models exposed in vitro and in vivo to a variety of spliceosome modulators alone or in combination with ATRi. Finally, we will perform a genome-wide CRISPR/Cas9 screen to identify genes and pathways that could sensitize spliceosome-mutant cells to the cytotoxic effects of ATRi that could be pursued in future clinical trials. This project addresses SPORE translational endpoints by conducting a first in human clinical trial of an ATRi in patients with myeloid malignancies. We will identify genetic biomarkers that are predictive of response to ATRi and establish the preclinical data necessary to nominate combination therapies that could be used to develop the first novel:novel combination of drugs for the treatment of MDS and AML with splicing factor mutations.